A major molecular hallmark of cancer cells is the aberrant splicing of genes with antagonistic splice isoforms. One of such genes is the vascular endothelial growth factor-A (VEGF-A) that can be spliced as either canonical pro-angiogenic isoforms or anti-angiogenic isoforms. In most normal adult tissues, VEGF-A is predominantly spliced as the anti-angiogenic b isoforms, whereas the pro-angiogenic isoforms become dominant in cancer cells to promote the growth of new blood vessels. Since sustained angiogenesis is critical to tumor growth and metastasis, restoration of normal VEGF-A splicing of in tumors presents a new anti-tumor approach. Long-term objectives and specific aims: We propose to develop a novel anti-angiogenesis approach by manipulating VEGF-A splicing. Such method will rewire the natural splicing regulatory pathway that orchestrates angiogenesis, thus inhibiting tumor growth. There are two specific aims in this project: (1) Specifically modulate VEGF-A splicing to promote anti- angiogenic isoforms; (2) Determine the in vivo efficacy of VEGF-A splicing modulation as anti-angiogenic therapy. Research design and methods Using a new technique recently developed in our lab, we will generate novel engineered splicing factors (ESFs) to shift the splicing of VEGF-A from angiogenic isoforms to anti-angiogenic isoforms. The designer ESFs will be expressed in metastatic breast and lung cancer cells, and the induction of anti-angiogenic VEGF- A splicing isoforms will be measured at mRNA and protein levels. As a complementary approach, we will use antisense oligonucleotides (ASOs) to shift the VEGF-A splicing toward anti-antigenic isoforms. We will further examine the response of the VEGF-A downstream signaling pathway to such splicing modulation. In addition, we will investigate whether the splicing modulation leads to the inhibition of angiogenesis using a variety of cell-based assays, and test the off-target effects of splicing modulation. We will further examine if the modulation of VEGF-A splicing can inhibit the tumor angiogenesis and growth in xenograft mouse model of human cancers. We will inject cancer cells pretreated with ESFs or ASOs into SCID mice to determine if the splicing modulation can inhibit the xenograft tumor formation and growth. Further we will directly treat the established xenograft tumors with intratumoral injection or systematic delivery of ESF or ASOs, and determine if such treatment can inhibit tumor progression. We will also combine splicing modulation of VEGF-A with other cancer therapies to see if they have synergistic effect to inhibit tumor progression in live animals.